REGULATION OF SITE-SPECIFIC RECOMBINATION BY SITE-SPECIFIC RECOMBINASE/NUCLEAR RECEPTOR FUSION PROTEINS

Abstract: An invention is described that permits the regulation of recombination in cells, organisms or appropriate cell-free systems. The invention involves creating fusion proteins between recombinase proteins, or components of recombinase systems, and ligand binding domains derived from nuclear receptors. The fusion proteins show little recombinase activity in the absence of the ligand that binds to the ligand binding domain. Upon binding of the ligand, recombinase activity is induced. The invention provides a practical means to regulate recombination in cells and organisms and, by linking ligand binding to recombination, provides a simple means whereby ligand binding can be measured as recombination achieved.

Description: Regulation of site-specific recombination by site-specific recombinase/nuclear receptor fusion proteins Specification The present invention relates to the technical field of genetic manipulation, and more specifically, to the use of recombina¬ tion-mediated DNA rearrangements and to the use of the regula¬ tory potential of nuclear receptors. The use of site-specific recombinases (SSRs) to induce defined rearrangements of DNA has been described in a variety of organ¬ isms (1-12) . These reports describe the introduction of a DNA construct that contains SSR target sites. Subsequent exposure to the SSR enzyme activity resulted in the DNA rearrangement determined by the disposition of the target sites (see refe¬ rence 13 for a recent review of SSRs) . Three SSRs have been used in this manner to date; FLP recombinase from the 2μ epi- some of Saccharomyces cerevisiae (1,2,5,6,9,10), CRE recombi¬ nase from the Escherichia coli phage Pl (3,4,8,11,12) and R recombinase from pSRl of Zygosaccharomyces rouxii (7) . Amongst other SSR systems relevant to the invention described here are those listed in references 13 and 14, and SSRs from Kluyveromy- ces drosophilarium (15) , Kluyveromyces waltii (16) , λ Int (17) and the Gin recombination system from phage Mu (18) . The con¬ tent of the above document is incorporated by reference. For many applications in cells, organisms and cell-free in vitro systems, SSR induced DNA rearrangements must be regula¬ ted. Current implementation of the potential offered by SSRs is limited by the means available to regulate SSR activity. In experiments with cultured cells, unregulated SSRs have been used. For example, after introduction of SSR target sites into cells, recombination has been induced by subsequent introduc¬ tion of either FLP recombinase by transfection of DNA (4,9) or injection of CRE recombinase protein (12) . That is, the inten¬ ded recombination event was regulated merely by the time of introduction of an appropriate macromolecule. Amongst other limitations, this precludes the creation and proliferation of homogeneous populations of cells that contain both the unrear- ranged target sites and the SSR and in which the recombination event can be induced after cell numbers have been expanded. In experiments with transgenic animals, the issue of SSR regu¬ lation has been addressed by regulating the expression of an introduced SSR gene using the inducible heat-shock promoter in Drosophila (5) or a tissue-specific promoter in mice (11) . Both approaches have limited applicability. Namely, heat-shock regu¬ lation of transgene expression is currently only useful in flies and no suitable counterpart is available for use in cell lines or vertebrates. Also, the use of a tissue-specific promo¬ ter to regulate transgene expression relies on the limited availability of suitable promoters and enhancers and the ex¬ pression pattern achieved is confined to the times and places at which these tissue specific elements are active. The problem underlying the present invention was to provide a regulated recombination system, wherein the disadvantages of the prior art are at least partially eliminated. More specifi¬ cally, the problem was to provide a recombination system, wherein the recombination event can be induced independently from any tissue specific restrictions. This patent application describes an invention that regulates SSR activity, rather than its expression. Thus any means of achieving and directing expression can be used, such as using ubiquitous or broadly active promoters and enhancers, as well as tissue specific or inducible promoters and enhancers. One aspect of the present invention relates to a fusion pro¬ tein, comprising a recombinase protein or a component of a re¬ combinase complex, fused to part or all of a nuclear receptor, so that the amino acids that bind the ligand of said nuclear receptor are included, such that in cells or appropriate cell- free systems: (a) recombinase activity is inhibited in the ab¬ sence of ligand binding to said ligand binding domain and (b) recombinase activity is induced or altered by binding of ligand to said ligand binding domain. oo 00 © & H U P, ro IΛ 8 00 00 o 63 H u ^f •0 8 00 e 00 © H α PH χ χ> 9\ 00 o 00 © H U ~ n £ gen receptor. The plasmid pHFEl has been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) , Mascheroder eg lb, D-38124 Braunschweig on June 25, 1994 under the accession number DSM 9265 according to the requirements of the Budapest Treaty. Still a further subject-matter of the present invention is a cell containing a nucleic acid or a recombinant vector as defi¬ ned above. Preferably, the cell is stably transformed with the nucleic acid or the vector. Suitable cells are eukaryotic or prokaryotic cells. Examples of prokaryotic cells are gram-nega¬ tive bacterial cells, especially E.coli cells. Examples of eukaryotic cells are mammalian cells, yeasts and trypanosomes. The invention separates the function of ligand binding from the functions of transcription factors, coupling ligand binding to recombinase activity. Thereby, the binding of ligand can be assessed by any means that measures recombinase activity. Thus, further embodiments of the invention include methods for deter¬ mining the binding of ligand to the LBD of a nuclear receptor, comprising the steps of: (i) the introduction of the SSR-LBD fusion protein or the nu¬ cleic acid coding therefor into cells, or appropriate cell-free systems, that contain the DNA target sites for the SSR; (ii) administering the ligand or a mixture suspected to contain a ligand or ligands to be evaluated; if the ligand is not already present in the system; (iii) detecting the recombinase activi¬ ty, if any, of the SSR-LBD by detecting, directly or indirect¬ ly, recombination or changes in the recombination rate between the DNA target sites. The introduction of SSR-LBD fusion proteins into cells may be accomplished by transforming the cells with appropriate vectors containing a nucleic acid coding for the SSR-LBD fusion pro¬ tein. The administration of the ligand to be evaluated is not necessary in systems which already contain the ligand and in which the ligand-concentration is determined by detecting re¬ combination between DNA target sites. 00 e 00 υ H PH cn $ (c) effecting a recombination of said DNA target sites by con¬ tacting the fusion protein with a ligand for the nuclear recep¬ tor component of the fusion protein, e.g. by adding the ligand to the system at a predetermined time, or by effecting produc¬ tion of the ligand in the system at a predetermined time. Description of the Figures Figure IA depicts the plasmid, pNEOSGAL (thin line) , integrated into the genome of 293 cells (thick line) . FLP recombinase tar¬ get sites (FRTs) are depicted as broad arrows and the neomycin resistance gene lies between the two FRTs. Upon estradiol admi¬ nistration, the DNA between the FRTs is excised, leaving one FRT in the genome and one on the excised circular DNA. The probe used for the Southern blot of Fig. 2 is also depicted. Figure IB depicts the steps involved in the creation of plasmid pHFEl . Figure 2 shows a Southern blot of a timecourse of recombination occurring in the cell line P1.4. At the times indicated after estradiol, or ethanol only, administration in the upper part of the figure, cells were lysed, DNA purified, restricted with Ndel, run on a 1.25% agarose gel and blotted to Biodyne B mem¬ branes by standard methodologies. The membrane was hybridized with the radioactively labelled probe depicted in Fig. IA. The upper band shows the unrecombined integrated DNA and the lower the recombined band. Estradiol was dissolved in ethanol and the extreme right hand lane shows cells treated with the equivalent amount of ethanol, without estradiol, for 51 hours. Figure 3 shows the complete amino acid sequence of the SSR-LBD- fusion polypeptide encoded by the plasmid pHFEl. The FLP recom¬ binase domain is located from amino acid 1-423. The linker peptide is located from amino acid 424-428. The estrogen bin¬ ding domain is located from amino acid 429-773. Figure 4 shows the nucleic acid sequence coding for the SSR-LBD fusion polypeptide of Figure 3. The parts coding for the FLP recombinase domain, the linker peptide and the estrogen binding domain, respectively, are specified. The following examples are offered by way of illustration and is not intended to limit the invention in any manner. Example 1 Construction of plasmid containing a SSR-LBD-fusion gene The experiment involves two plasmids, one of which is the tar¬ get for recombination, and the other is the recombinase expres¬ sion plasmid. As the target plasmid, pNEOSGAL (9) was employed. pNEOEGAL contains two FLP recombinase target sites (FRT) sur¬ rounding a gene encoding neomycin resistance (Fig. la) . The expression plasmid, pHFE 1, carries two genes, one encoding an FLP recombinase-estrogen receptor LBD fusion protein and the other encoding hygromycin resistance (Fig. lb) . The plasmid was constructed by standard cloning procedures using pBluescribe (Stratagene) , pOG 44 (9) , pHE63 (22) and pCNH2 (27) . The stop codon of the FLP recombinase coding region present in pOG44 was mutated by oligonucleotide replacement to introduce BamHI, BsiWl and EcoRI sites and continue the open reading frame. The BamHI site in the estrogen receptor encoded by pHE63 was used to join the coding regions of FLP recombinase and estrogen receptor LBD. All of the estrogen receptor coding region car¬ boxy to the BamHI site is present in the plasmid, including its stop codon. Example 2 Regulation of site specific recombination pNEOSGAL was introduced into 293 human embryonal kidney cells by electroporation (5 x 10 6 cells in 500 μl phosphate-buffered saline with 5 μg of Apal linearised plasmid; 300 V at 125 μF in a 1 ml cuvette using a Bio-Rad Gene Pulser) and selection for neomycin resistance (G418, 0.4 mg/ml) . Single colonies of resi¬ stant cells were cloned and characterised for incorporation of target plasmid DNA by Southern blotting. A clone, Pl, showing integration of a single copy of target plasmid DNA was selected and transfected, using the calcium phosphate procedure, with the second plasmid, pHFE 1 (1 μg of XmnI linearised pHFE 1 was precipitated by the standard calcium phosphate method onto IO 7 Pl cells which were cultured in DMEM without phenol red, 10% charcoal stripped fetal calf serum) . Four clones resistant to both neomycin and hygromycin (0.4 mg/ml G418, 0.4 mg/ml hygro- mycin B) were isolated and the dependance of recombination on estradiol administration was observed in all four. For clone P1.4, a time course of recombination in the presence of IO "6 M estradiol is shown (Fig. 2) . References 1. FalccS.C, Li,Y., BroachJ.R. and Botstein,D. (1982) Cell 29, 573-584. 2. Cox,M.M. (1983) Proc. Nad. Acad. Sci. USA 80, 4223-4227. 3. Sauer,B. (1987) Mol. Cell. Biol. 7, 2087-2096. 4. Sauer,B. and Henderson,N. (1989) Nucleic Acids Res. 17,147-161. 5. Golic,K.G. and Lindquist,S. (1989) Cell 59, 499-509. 6. Cregg,J.M. and Madden,K.R. (1989) Mol. Gen. Genet. 219, 320-323. 7. Matsuzaki,H. et al, (1990) J. Bacteriol. 172, 610-618. 8. OdellJ., Caimi,P., Sauer,B. and RussellS. (1990) Mol. Gen. Genet 223, 369-378. 9. O'Gorman,S., Fox,D.T. and WahUG.M. (1991) Science 251, 1351-1355. 10. Konsola i,M. et al (1992) New Biologist 4, 551-557. 11. Lakso,M. et al (1992).Proc. Natl. Acad. Sci. USA 89, 6232-6236. 12. BaubonisΛV. and Sauer,B. (1993) Nucleic Acids Res. 21,2025-2029. 13. Stark V.M. et al (1992) Trends Genet. 8, 432-439. 14. Utatsu . et al (1987) J. Bacteriol. 169, 5537-5545. 15. ChenXJ. et al (1986) Nucleic Acids Res. 14, 4471-4481. 16. ChenXJ. et al (1992) J. Gen. Microbiol. 138, 337-345. 17.Landy,A. (1989) Annu. Rev. Biochem. 58, 913-949. 18. Klippel.A. et l (1993) EMBO J. 12, 1047-1057. 19. Webster,N.J.G. et al (1988) Cell 54, 194-207 20. Beato,M. (1989) Cell 56, 335-344. 21. Kumar,V. et al (1987) Cell 51, 941-951. 22. Picard,D., Salser,S. and Yamamoto,K.R. (1988) Cell 54, 1073-1080. 23. Eilers,M., Picard,D., YamamotoXR. and Bishop.M. (1989) Nature 340, 66-68. 24. Supeπi-Furga,G. et al (1991) Proc. Nad. Acad. Sci. USA 88, 5114-5118. 25. Burk,O. and KlempenauerX-H. (1991) EMBO J. 10, 3713-3719. 26. Boehmelt,G. et al (1992) EMBO J. 11, 4641-4652. 27. Schraidt,E. et al (1990) Mol. Cell. Biol. 10, 4406-4411.

Claims: Claims 1. A fusion protein, comprising a recombinase protein or a component of a recombinase complex, fused to part or all of a nuclear receptor so that the amino acids that bind the ligand of said nuclear receptor are included, such that, in cells or appropriate cell-free systems: (a) recombinase activity is inhibited in the absence of ligand binding to said ligand binding domain, and (b) recombinase activity is induced or altered by binding of ligand to said ligand binding domain. 2. The fusion protein of claim 1, wherein the nuclear receptor is a hormone receptor. 3. The fusion protein of claim 1 or 2, wherein the nuclear receptor is a steroid hormone receptor. 4. The fusion protein of any one of the claims 1-3, wherein the nuclear receptor is a mutated derivative of a nuclear receptor such that it retains the characteristics of the fusion protein of claim 1. 5. The fusion protein of any of the claims 1 - 4, wherein the nuclear receptor is a vertebrate glucocorticoid, estrogen, progesteron or androgen receptor. 6. The fusion protein of any one of the claims 1-5, wherein the recombinase protein or component of a recombi¬ nase complex is selected from the group consisting of: FLP recombinase, Cre recombinase, R recombinase from the Zygo- saccharomyces rouxii plasmid pSRl, A recombinase from the Kluyveromyces drosophilarium plasmid pKDl, A recombinase from the Kluyveromyces waltii plasmid pK l, any component of the λ Int recombination system, any component of the Gin recombination system, or a variant thereof. 7. The fusion protein of any of claims 1-6, wherein the recombinase protein or compound of a recombinase complex is fused to the nuclear receptor or ligand binding domain thereof by means of a genetic fusion. 8. The fusion protein of any of claims 1-7, wherein said nuclear receptor or ligand binding domain the¬ reof is fused to the N- or C-terminus of said recombinase protein or component of a recombinase complex. 9. The fusion protein of any of claims 1-8, wherein said nuclear receptor is fused to said recombinase protein or component of a recombinase complex through a peptide linker. 10. The fusion protein of any of claims 1-9, which comprises the FLP recombinase and the ligand binding domain of the estrogen receptor. 11. A nucleic acid which encodes the fusion protein of any one of the claims 1-10. 12. The nucleic acid of claim 11, which is a DNA or RNA. 13. A recombinant vector comprising at least one copy of the nucleic acid of claims 11 or 12. 14. The vector of claim 13, which is a plasmid. 15. The plasmid pHFEl (DSM 9265) 16. A cell containing a nucleic acid of claims 11 or 12 or a recombinant vector of any one of the claims 13-15. 17. The cell of claim 16, which is a mammalian cell. 18. A method for determining the binding of a ligand to the ligand binding domain of a nuclear receptor, comprising the steps of: (a) the introduction of the fusion protein of claims 1-9, or the nucleic acid of claims 10 or 11 into cells, or appropri¬ ate cell-free systems, that contain the DNA target sites for recombination, (b) optionally administering the ligand or a mixture suspec¬ ted to contain a ligand or ligands, to be evaluated, (c) detecting the recombinase activity, if any, of said fusion protein by detecting recombination or changes in the recombination rate between said DNA target sites. 19. The method of claim 18, wherein the recombination between said DNA target sites is detected by direct measurement of the physical arrangement of said target sites. 20. The method of claim 18, wherein the recombination between said DNA target sites is detected by assessing the properties encoded by the DNA regions carrying the target sites before or after recombina¬ tion. 21. A method for determining the effect of mutations in the ligand binding domain of a nuclear receptor on its ability to bind ligand, comprising the steps of: (a) introducing mutations into the ligand binding domain of the fusion protein of claims 1-10, (b) following steps (a) to (c) of claim 18. 22. A method for regulating the recombination of DNA target sites, comprising the steps of: (a) providing cells or appropriate cell-free systems that contain DNA target sites for a site specific recombinase, (b) contacting the DNA target sites with a fusion protein according to any one of the claims 1-10, and (c) effecting recombination of said DNA target site by con¬ tacting the fusion protein with a ligand for the nuclear receptor component of said fusion protein.

Inventors: STEWART FRANCIS, STEWART, FRANCIS
Applicants: EUROP MOLECULAR BIOLOGY LAB EM, STEWART FRANCIS, EUROPEAN MOLECULAR BIOLOGY LABORATORY (EMBL), STEWART, FRANCIS
Application year: 1994
Main classification: C07K15
IPC codes: C07K14, C07K19, C12N5, C12N9, C12N15